Distinct Characteristics of Transcellular Transport between Nicotine and Tetraethylammonium in LLC-PK 1 Cells 1

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1 /98/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 2 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286: , 1998 Distinct Characteristics of Transcellular Transport between Nicotine and Tetraethylammonium in LLC-PK 1 Cells 1 KENJI TAKAMI, HIDEYUKI SAITO, MASAHIRO OKUDA, MIKIHISA TAKANO and KEN-ICHI INUI Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto , Japan Accepted for publication April 7, 1998 This paper is available online at ABSTRACT To clarify the mechanisms of the renal tubular secretion of nicotine, we studied transport of nicotine in the kidney epithelial cell line LLC-PK 1. The transcellular transport of nicotine from the basolateral side to the apical side of the LLC-PK 1 monolayers grown on membrane filters was much greater than that of tetraethylammonium. The basolateral-to-apical transport of nicotine was stimulated by lowering the ph of the apical side, accompanied by a decrease in the accumulation of nicotine. The accumulation of nicotine from the basolateral side was inhibited by unlabeled nicotine, cotinine, tetraethylammonium, cimetidine and quinidine. The uptake of nicotine across the Nicotine is consumed widely by tobacco smokers and in therapeutic pharmaceuticals such as nicotine-resin chewing gum. Nicotine consists of a pyridine and a pyrrolidine ring, thereby being a weak base (pk a 7.9) (Beckett et al., 1972). The major elimination route of nicotine is its extensive metabolism in the liver (Kyerematen and Vesell, 1991; Plowchalk et al., 1992). In addition to the hepatic metabolism, both the parent nicotine and its metabolites are excreted into the urine not only by glomerular filtration but also by active tubular secretion (Svensson, 1987). Nicotine is excreted in urine in a ph-dependent manner (Benowitz and Jacob III, 1985; Rosenberg et al., 1980). At urinary ph levels of 7, nicotine is reabsorbed by the renal tubules and only 2% of a dose is excreted unchanged in urine. At urinary ph levels of 5, 23% of the nicotine dose is recovered in urine (Rosenberg et al., 1980). Although this phdependent renal handling of nicotine has been thought to follow the predictions of the Henderson-Hasselbach equation, the precise mechanisms involved in the tubular secretion and reabsorption of nicotine have not yet been fully characterized. Nicotine has been reported to interact with an organic cation transport system in renal tubules (Bendayan et al., 1990a; Ullrich et al., 1993; Wong et al., 1991). Renal nicotine clearance was decreased by cimetidine, an organic cation (Bendayan et Received for publication November 28, This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, and Culture of Japan and by the Smoking Research Foundation. ABBREVIATION: PCMBS, p-chloromercuribenzene sulfonate. 676 apical membrane was inhibited by unlabeled nicotine and quinidine but not by tetraethylammonium or cimetidine. Pretreatment with p-chloromercuribenzene sulfonate caused a decrease in the transcellular transport of tetraethylammonium but not of nicotine. These results suggest that nicotine undergoes vectorial transport from basolateral side to the apical side of LLC-PK 1 monolayers in a H gradient-dependent manner, corresponding to the secretion in the renal tubules. Nicotine transport in LLC-PK 1 cells could be mediated by a transport system that is distinct from the transport system for tetraethylammonium. al., 1990b), but it is unknown whether the organic cation transport systems expressed in the tubular cells are involved in the renal secretion of nicotine. LLC-PK 1 cells, derived from the pig kidney, have been used for studies of the transepithelial transport and accumulation of cationic molecules and drug interactions (Bendayan et al., 1994; McKinney et al., 1992; Ohtomo et al., 1996; Saito et al., 1992). We obtained the first evidence that the apical membranes of the LLC-PK 1 cells express the H /organic cation antiporter (Inui et al., 1985). In addition, we reported that LLC-PK 1 cell monolayers grown on porous membrane filters showed a unidirectional transcellular transport of the typical cation tetraethylammonium from the basolateral side to the apical side, corresponding to the secretion in the renal proximal tubules (Saito et al., 1992). In the present study, we investigated the mechanisms underlying the transcellular transport of nicotine in the LLC- PK 1 cell monolayers, and we obtained the first evidence showing that the H gradient-dependent transcellular transport of nicotine is mediated by apically and basolaterally localized organic cation transport systems, which are distinct from the transport systems for tetraethylammonium in the LLC-PK 1 cells. Experimental Procedures Materials. [ 14 C]Tetraethylammonium bromide (124 MBq/mmol), ( )-[ 3 H]nicotine (2,900 GBq/mmol), D-[ 3 H]mannitol (729 GBq/mmol)

2 1998 Transport of Nicotine in Renal Cells 677 and D-[ 14 C]mannitol (1.9 GBq/mmol) were purchased from DuPont- New England Nuclear Research Products (Boston, MA). Nicotine was obtained from Wako Pure Chemical Industries (Osaka, Japan). Cotinine and N 1 -methylnicotinamide were purchased from Sigma Chemical (St. Louis, MO). Tetraethylammonium chloride, cimetidine, quinidine sulfate and PCMBS were from Nacalai Tesque (Kyoto, Japan). Levofloxacin was kindly supplied by Daiichi Seiyaku (Tokyo, Japan). All other chemicals were of the highest purity available. Cell culture. Cells of the porcine kidney epithelial cell line LLC- PK 1, obtained from the American Type Culture Collection (ATCC CRL-1392; Rockville, MD), were grown on plastic dishes in Dulbecco s modified Eagle s medium (GIBCO Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum (Whittaker Bioproducts, Walkersville, MD) without antibiotics in an atmosphere of 5% CO 2 /95% air at 37 C (Saito et al., 1992). For the transport experiments, the cells were seeded on microporous membrane filters (3- m pores, 4.71-cm 2 growth area) inside a Transwell cell culture chamber (Costar, Cambridge, MA) at a cell density of cells/ cm 2. In this study, LLC-PK 1 cells between the 212th and 220th passages were used. Measurements of transcellular transport and cellular accumulation of nicotine and tetraethylammonium. The transcellular transport and cellular accumulation of radioactive drugs were measured in monolayer cultures grown in the Transwell chamber. The incubation medium for uptake experiments was Dulbecco s phosphate-buffered saline (ph 7.4) (buffer: 137 mm NaCl, 3 mm KCl, 8mMNa 2 HPO 4, 1.5 mm KH 2 PO 4, 1 mm CaCl 2 and 0.5 mm MgCl 2 ), containing 5 mm D-glucose. In the general experiments, the transcellular transport and cellular accumulation were measured as described previously (Saito et al., 1992). Briefly, after the removal of culture medium from both sides of the monolayers, the cell monolayers preincubated with 2 ml of incubation medium containing [ 3 H]nicotine (12.5 or 25 nm, 37 or 74 kbq/ml) or [ 14 C]tetraethylammonium (50 M, 6.3 kbq/ml) and D-[ 14 C]mannitol (3.9 M, 7.4 kbq/ ml) or D-[ 3 H]mannitol (6.4 M, 29.6 kbq/ml) were added to the basolateral or apical side of the monolayers, and 2 ml of radioactivity-free incubation medium was added to the opposite side. The cell monolayers were incubated for the specified periods of time at 37 C. Labeled D-mannitol was used to estimate the paracellular fluxes and the extracellular trapping of labeled drugs. For the transport measurements, an aliquot (50 l) of the incubation medium in the other side was taken at the specified time, and the radioactivity was determined. For accumulation measurements, the medium was removed by suction at the end of the incubation period, and the monolayers were rapidly washed two times with 2 ml of ice-cold incubation medium on each side. The filters with monolayers were detached from the chambers, the cells on the filters were solubilized in 0.5 ml of 1 N NaOH, and the radioactivity of each aliquot (200 l) was determined. The radioactivity of the collected media and the solubilized cell monolayers were determined in 5 ml of ACS II (Amersham International, Buckinghamshire, UK) by liquid scintillation counting. The protein content of the solubilized cell monolayers was determined by the method of Bradford (Bradford, 1976), using the BioRad Protein Assay Kit with bovine -globulin as the standard. The protein content of the monolayers was 0.94 to 1.45 mg/filter (4.71 cm 2 ). Statistical analysis. Data were analyzed using one-way analysis of variance followed by Fisher s t test. Probability values of 5% were considered significant. LLC-PK 1 cells grown on filters in a Transwell chamber in the presence of an inward H gradient (apical side, ph 6.0; basolateral side, ph 7.4). As shown in figure 1, the basolateral-to-apical transports of [ 14 C]tetraethylammonium and [ 3 H]nicotine were much higher than the apical-to-basolateral transport of each drug, suggesting that both drugs were subjected to unidirectional transcellular transport in the LLC-PK 1 cells, corresponding to the renal tubular secretion. The cellular accumulation of [ 14 C]tetraethylammonium from the basolateral side for 60 min was 3-fold higher than that from the apical side (fig. 1B). In contrast, the accumulation of [ 3 H]nicotine from the basolateral side was smaller than that from the apical side (fig. 1D), suggesting that the uptake of nicotine occurred across the apical membranes of the LLC- PK 1 cells. Effect of the apical side ph. It is well known that the renal excretion of nicotine is urine ph dependent (Rosenberg et al., 1980). When urine becomes more acidic (ph 5), as much as 23% of a dose can be recovered unchanged in the urine. To determine whether this ph-dependent manner of nicotine secretion in vivo would take place in LLC-PK 1 cells, we examined the effect of ph of the incubation medium on the transcellular transport and accumulation of nicotine. Figure 2 illustrates the ph dependence of the [ 3 H]nicotine transport and accumulation. The basolateral-to-apical transport of [ 3 H]nicotine was markedly increased by lowering the ph of the apical side (the basolateral side ph was fixed to 7.4), accompanied by a decrease in the accumulation in the monolayers. These results suggested that the transcellular transport of nicotine is dependent mainly on the ph of the apical side and that the antiport of nicotine with H is involved in the efflux of nicotine out of LLC-PK 1 cells. Concentration dependence of nicotine accumulation. Figure 3 shows the cellular accumulation of [ 3 H]nicotine and [ 14 C]tetraethylammonium from the basolateral side of the LLC-PK 1 cell monolayers as a function of an increased concentration of the substrate. The curves for the accumulation of both drugs were curvilinear, indicating a saturable process. The apparent Michaelis constant (K m ) and maximum velocity (V max ) values of the accumulation of [ 3 H]nicotine, estimated from the Michaelis-Menten equation using a Results Transcellular transport and cellular accumulation of tetraethylammonium and nicotine. We measured the transcellular transport and cellular accumulation of [ 14 C]tetraethylammonium and [ 3 H]nicotine by the monolayers of Fig. 1. Transcellular transport and accumulation of [ 14 C]tetraethylammonium (A and B) and [ 3 H]nicotine (C and D) by LLC-PK 1 cell monolayers. LLC-PK 1 cell monolayers were incubated at 37 C with 2 ml of 50 M [ 14 C]tetraethylammonium or 25 nm [ 3 H]nicotine added to the basolateral (E, ph 7.4) or apical (, ph6.0) side of the monolayers. Appearance of radioactivity in the opposite side (2 ml) was periodically measured. After a 60-min incubation, the radioactivity of solubilized cells was counted. Each point or column represents the mean S.E. of three monolayers.

3 678 Takami et al. Vol. 286 Fig. 2. Effect of the ph of the apical side on the transcellular transport (A) and accumulation (B) of [ 3 H]nicotine by LLC-PK 1 cell monolayers. LLC-PK 1 cell monolayers were incubated at 37 C for 15 min, with 2 ml of 12.5 nm [ 3 H]nicotine (ph 7.4) added to the basolateral side and with various ph levels on the apical side. The appearance of radioactivity in the apical side and accumulation were measured. Each point represents the mean S.E. of three monolayers. Fig. 3. Concentration dependence of the accumulation of [ 14 C]tetraethylammonium (A) and [ 3 H]nicotine (B) by LLC-PK 1 cell monolayers. LLC- PK 1 cell monolayers were incubated at 37 C for 5 min with 2 ml of varying concentrations of [ 14 C]tetraethylammonium and [ 3 H]nicotine (ph 7.4) added to the basolateral side. Accumulation was measured at ph 7.4 of the apical side. The dashed line and the curve ( ) represent the nonsaturable component and the specific component of the nicotine accumulation, respectively. Each point represents the mean S.E. of three monolayers. nonlinear least-squares analysis (Yamaoka et al., 1981), were 360 M and 916 pmol/mg of protein/min, respectively. The K m and V max values for [ 14 C]tetraethylammonium were 296 M and 592 pmol/mg of protein/min, respectively. The K d value (a parameter for the diffusion component) of [ 14 C]tetraethylammonium was negligible, whereas the K d value of [ 3 H]nicotine was 206 pmol/mg of protein/min/mm. Effects of cationic drugs on tetraethylammonium and nicotine accumulation. To examine the substrate specificity of the transport systems mediating the cellular accumulation of nicotine and/or tetraethylammonium in LLC-PK 1 cells, we evaluated the effects of various cationic compounds added to the basolateral or apical side on the accumulation of [ 14 C]tetraethylammonium and [ 3 H]nicotine from the basolateral and apical sides of the monolayers. As depicted in figure 4, cationic drugs such as unlabeled nicotine, tetraethylammonium, cimetidine and quinidine showed potent inhibitory effects on the accumulation of both drugs from the basolateral side. N 1 -Methylnicotinamide, an endogenous cation, had no significant effect on the accumulation. Cotinine, a major metabolite of nicotine, showed a potent inhibitory effect on the accumulation of [ 3 H]nicotine but not on that of [ 14 C]tetraethylammonium. In contrast, the [ 14 C]tetraethylammonium accumulation from the apical side Fig. 4. Effect of cationic drugs on the accumulation of [ 14 C]tetraethylammonium (A) and [ 3 H]nicotine (B) from the basolateral side by LLC-PK 1 cell monolayers. LLC-PK 1 cell monolayers were incubated at 37 C for 15 minwith2mlof50 M [ 14 C]tetraethylammonium or 12.5 nm [ 3 H]nicotine (ph 7.4) added to the basolateral side in the absence (control) and presence of the other cationic drug (1 mm) on the same side. Accumulation was measured at ph 6.0 of the apical side. Each column represents the mean S.E. of three monolayers., P.01, significant differences from the control using analysis of variance followed by Fisher s t test. was potently inhibited in the presence of nicotine and quinidine, and weakly but significantly inhibited in the presence of unlabeled tetraethylammonium and cimetidine (fig. 5). Furthermore, the [ 3 H]nicotine accumulation from the apical side was markedly inhibited in the presence of unlabeled nicotine or quinidine, whereas the accumulation was not inhibited in the presence of unlabeled tetraethylammonium, cimetidine or N 1 -methylnicotinamide. These results suggested that the substrate specificity of the transport system, which mediates the tetraethylammonium transport in the basolateral and apical membranes, differs from that of the systems mediating the nicotine transport in LLC-PK 1 cells. Effects of PCMBS on transcellular transport of tetraethylammonium and nicotine. We previously reported Fig. 5. Effect of cationic drugs on the accumulation of [ 14 C]tetraethylammonium (A) and [ 3 H]nicotine (B) from the apical side by LLC-PK 1 cell monolayers. LLC-PK 1 cell monolayers were incubated at 37 C for 15 min with2mlof50 M [ 14 C]tetraethylammonium or 12.5 nm [ 3 H]nicotine (ph 7.4) added to the apical side in the absence (control) and presence of the other cationic drug (1 mm) on the same side. Accumulation was measured at ph 7.4 of the basolateral side. Each column represents the mean S.E. of three monolayers., P.05;, P.01, significant differences from the control using analysis of variance followed by Fisher s t test.

4 1998 Transport of Nicotine in Renal Cells 679 that sulfhydryl groups are essential for the organic cation transport systems expressed in both the apical and basolateral membranes of LLC-PK 1 cells (Saito et al., 1992). In this study, we examined the effect of PCMBS on the transcellular transport and accumulation of [ 14 C]tetraethylammonium and [ 3 H]nicotine. As illustrated in figure 6, the apical pretreatment with PCMBS caused a marked decrease in the basolateral-to-apical transport of [ 14 C]tetraethylammonium, which was accompanied by an increase in the cellular accumulation. The pretreatment of the basolateral membranes with PCMBS had weak and not significant effect on the [ 14 C]tetraethylammonium transport and accumulation. In contrast, the transcellular transport and accumulation of [ 3 H]nicotine were not affected by the PCMBS pretreatment of the basolateral and apical membranes, suggesting that the transport systems for nicotine are almost insensitive to sulfhydryl modification. Discussion To clarify the mechanisms of the renal tubular secretion of nicotine, we examined the transcellular transport and cellular accumulation of nicotine by using cell monolayers of the porcine kidney epithelial cell line LLC-PK 1. It has been well documented that nicotine is both reabsorbed and secreted in the human renal tubules (Rosenberg et al., 1980; Svensson, 1987), although processes involved in the nicotine transport through renal basolateral and brush-border membranes have not yet been characterized. We previously reported that the LLC-PK 1 cells express organic cation transporters in both the basolateral and apical membranes and that tetraethylammonium was transported unidirectionally from the basolateral side to the apical side across monolayers grown on porous membrane filters (Saito et al., 1992). The apically localized H /organic cation antiporter was found to mediate the TEA efflux out of the cells in the apical membranes (Inui et al., 1985). Fig. 6. Effect of PCMBS on the transcellular transport and accumulation of [ 14 C]tetraethylammonium (A and B) and [ 3 H]nicotine (C and D) by LLC-PK 1 cell monolayers. LLC-PK 1 cell monolayers were preincubated at 4 C for 10 min with PCMBS added to the basolateral or apical side or to both sides of monolayers. After the removal of preincubation medium containing 0.1 mm PCMBS, the monolayers were washed once and incubated at 37 C for 60 min with 2 ml of 50 M[ 14 C]tetraethylammonium or 12.5 nm [ 3 H]nicotine (ph 7.4) added to the basolateral side. The appearance of radioactivity in the apical side (ph 7.4) and accumulation were measured. Each column represents the mean S.E. of three monolayers., P.01, significant differences from the control using analysis of variance followed by Fisher s t test. In the present study, we observed that both tetraethylammonium and nicotine were transported directionally from the basolateral side to the apical side in the presence of an inward H gradient on the apical membranes, corresponding to renal tubular secretion (fig. 1). The ph-dependent transcellular transport of nicotine is consistent with the urine ph-dependent excretion of nicotine in the kidney (Rosenberg et al., 1980). The ph dependence of the nicotine transport across LLC-PK 1 cell monolayers could be explained by the following possibilities. First, nicotine is taken up into the monolayer across the basolateral membrane and transported across the apical membrane via the H /organic cation antiport system; an imposed H gradient could drive the efflux of intracellular nicotine out of the cells, as has been observed for tetraethylammonium transport. Second, nicotine follows the Henderson-Hasselbach equation based on a ph partition theory. This possibility can be supported by the finding that ionized nicotine with lower permeability is increased by the lowering ph of the apical side, thereby resulting in an accelerated cell-to-apical passive diffusion of nonionized nicotine. This would not be the case for tetraethylammonium because tetraethylammonium exists mostly in a cationic form with the pk a value of 11 and is unable to easily permeate cell membranes due to its low lipid solubility. However, the present findings that the apical uptake of nicotine was inhibited by the presence of unlabeled nicotine, quinidine and levofloxacin suggest that the apical transport of nicotine may be mediated by a specific transport system or systems rather than by passive diffusion. We previously reported that cimetidine may share a common transport system with tetraethylammonium in the rat renal membrane vesicles (Takano et al., 1985). In the present study, cimetidine inhibited the tetraethylammonium uptake at the basolateral and apical membranes in LLC-PK 1 monolayers. However, cimetidine had a weak inhibitory effect on the nicotine uptake from the basolateral membranes and no effect on that from apical membranes. Together with these findings, nicotine transport in apical and basolateral membranes of LLC-PK 1 cells could be mediated by distinct transport systems from those for tetraethylammonium and cimetidine. The most distinct feature of the transport characteristics between tetraethylammonium and nicotine was the PCMBS sensitivities. The PCMBS treatment of LLC-PK 1 monolayers caused a marked depression of tetraethylammonium transport, especially at the apical membranes, being consistent with the previous finding that the H /organic cation antiport system in the renal brush-border membranes was highly sensitive to PCMBS (Saito et al., 1992). In contrast, the nicotine transport was not affected by PCMBS (fig. 6), suggesting that the apical membrane transport system involved in nicotine efflux differs from the system that mediates tetraethylammonium efflux. It is as yet unknown whether the transport system mediating the transcellular transport of nicotine in LLC-PK 1 cells is an organic cation transport system or another type of specific transport system. In conclusion, nicotine was accumulated across the basolateral membrane via a specific transport system and underwent efflux across the apical membranes in a ph-dependent

5 680 Takami et al. Vol. 286 manner, corresponding to urine ph-dependent tubular excretion, and was mediated by a transport system distinct from the organic cation system for TEA in the LLC-PK 1 cells. To our knowledge, this is the first report describing cellular mechanisms of nicotine secretion in renal epithelia. In addition, LLC-PK 1 cells can serve an in vitro model for the prediction and evaluation of renal interactions between nicotine and a wide variety of cationic drugs. References Beckett AH, Gorrod JW and Jenner P (1972) A possible relation between pka 1 and lipid solubility and the amounts excreted in urine of some tobacco alkaloids given to man. J Pharm Pharmacol 24: Bendayan R, Lo B and Silverman M (1994) Characterization of cimetidine transport in LLC-PK 1 cells. J Am Soc Nephrol 5: Bendayan R, Sellers EM and Silverman M (1990a) Inhibition kinetics of cationic drugs on N 1 -methylnicotinamide uptake by brush border membrane vesicles from the dog kidney cortex. Can J Physiol Pharmacol 68: Bendayan R, Sullivan JT, Shaw C, Frecker RC and Sellers EM (1990b) Effect of cimetidine and ranitidine on the hepatic and renal elimination of nicotine in humans. Eur J Clin Pharmacol 38: Benowitz NL and Jacob P III (1985) Nicotine renal excretion rate influences nicotine intake during cigarette smoking. J Pharmacol Exp Ther 234: Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: Inui K, Saito H and Hori R (1985) H -Gradient-dependent active transport of tetraethylammonium cation in apical-membrane vesicles isolated from kidney epithelial cell line LLC-PK 1. Biochem J 227: Kyerematen GA and Vesell ES (1991) Metabolism of nicotine. Drug Metab Rev 23:3 41. McKinney TD, Scheller MB, Hosford M, Lesniak ME and Haseley TS (1992) Basolateral transport of tetraethylammonium by a clone of LLC-PK 1 cells. JAmSoc Nephrol 1: Ohtomo T, Saito H, Inotsume N, Yasuhara M and Inui K (1996) Transport of levofloxacin in a kidney epithelial cell line, LLC-PK 1 : Interaction with organic cation transporters in apical and basolateral membranes. J Pharmacol Exp Ther 276: Plowchalk DR, Andersen ME and Debethizy JD (1992) A physiologically based pharmacokinetic model for nicotine disposition in the Sprague-Dawley rat. Toxicol Appl Pharmacol 116: Rosenberg J, Benowitz NL, Jacob P and Wilson KM (1980) Disposition kinetics and effects of intravenous nicotine. Clin Pharmacol Ther 28: Saito H, Yamamoto M, Inui K and Hori R (1992) Transcellular transport of organic cation across monolayers of kidney epithelial cell line LLC-PK 1. Am J Physiol 262:C59 C66. Svensson CK (1987) Clinical pharmacokinetics of nicotine. Clin Pharmacokin 12: Takano M, Inui K, Okano T and Hori R (1985) Cimetidine transport in rat renal brush border and basolateral membrane vesicles. Life Sci 37: Ullrich KJ, Rumrich G, David C and Fritzsch G (1993) Bisubstrates: substances that interact with renal contraluminal organic anion and organic cation transport systems. Pflügers Arch 425: Wong LTY, Smyth DD and Sitar DS (1991) Interference with renal organic cation transport by ( )- and ( )-nicotine at concentrations documented in plasma of habitual tobacco smokers. J Pharmacol Exp Ther 261: Yamaoka K, Tanigawara Y, Nakagawa T and Uno T (1981) A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharmacobiodyn 4: Send reprint requests to: Ken-ichi Inui, Ph.D., Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto , Japan.